iC3b-opsonized apoptotic cells mediate a distinct anti-inflammatory response and transcriptional NF-kappaB-dependent blockade.

In recent years, it has become apparent that the removal of apoptotic cells by macrophages and DC is not only noninflammatory, but also immune-inhibitory, in most although not all circumstances. Complement may be involved in the uptake of apoptotic cells via direct binding of bridging factors in some physiological circumstances, by opsonization and engagement of the complement receptors. In the current study, we use a complement-dependent system of apoptotic cell clearance by human-derived macrophages and DC. Using a luciferase reporter gene and measuring immune response to non-opsonic zymosan, we show that iC3b-apoptotic cells induce NF-kappaB inhibition in response to zymosan and LPS at the nuclear translocation, transcriptional and post-transcriptional levels, leading to profound inhibition of proinflammatory cytokines. In addition, interaction with iC3b-opsonized apoptotic cells is characterized by macrophage secretion of IL-10 and lack of TGF-beta secretion. In conclusion, in cells with iC3b receptors, opsonized apoptotic cells mediate a distinct anti-inflammatory response and transcriptional NF-kappaB-dependent blockage.

Opsonization of apoptotic cells by autologous iC3b facilitates clearance by immature dendritic cells, down-regulates DR and CD86, and up-regulates CC chemokine receptor 7.

Immature dendritic cells (iDCs) do not mature after uptake of apoptotic cells and may play a role in the induction of peripheral tolerance to self antigens derived from apoptotic material. The integrins, alphavbeta3, alphavbeta5, and the scavenger receptor, CD36, have been shown to mediate uptake of apoptotic cells by iDCs. However, it is not known whether the complement system, also takes part in this process. In this study we investigated the ability of iDCs to bind to apoptotic cells opsonized by iC3b. Monocyte-derived dendritic cells were offered apoptotic Jurkat cells opsonized by autologous iC3b and labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanineperchlorate. A significant increase (P < 0.001) in the amount of cleared apoptotic cells was seen at low ratios. Despite increased efficiency of uptake, interaction between iC3b-opsonized apoptotic cells and iDCs down-regulated the expression of major histocompatibility complex class II, CD86, CC chemokine receptor (CCR)2, CCR5, and beta2-integrins (P < 0.001), and up-regulated expression of CCR7 (P < 0.001). In addition, iDC maturation responses to CD40L and lipopolysaccharide were significantly inhibited. We conclude that opsonization of apoptotic cells by iC3b induces tolerant iDCs that are able to migrate to lymph nodes.

Constitutive neutrophil apoptosis: regulation by cell concentration via S100 A8/9 and the MEK-ERK pathway.

Programmed cell death (PCD) is a fundamental mechanism in tissue and cell homeostasis. It was long suggested that apoptosis regulates the cell number in diverse cell populations; however no clear mechanism was shown. Neutrophils are the short-lived, first-line defense of innate immunity, with an estimated t = 1/2 of 8 hours and a high turnover rate. Here we first show that spontaneous neutrophil constitutive PCD is regulated by cell concentrations. Using a proteomic approach, we identified the S100 A8/9 complex, which constitutes roughly 40% of cytosolic protein in neutrophils, as mediating this effect. We further demonstrate that it regulates cell survival via a signaling mechanism involving MEK-ERK via TLR4 and CD11B/CD18. This mechanism is suggested to have a fine-tuning role in regulating the neutrophil number in bone marrow, peripheral blood, and inflammatory sites.

Thrombospondin-1-N-terminal domain induces a phagocytic state and thrombospondin-1-C-terminal domain induces a tolerizing phenotype in dendritic cells.

In our previous study, we have found that thrombospondin-1 (TSP-1) is synthesized de novo upon monocyte and neutrophil apoptosis, leading to a phagocytic and tolerizing phenotype of dendritic cells (DC), even prior to DC-apoptotic cell interaction. Interestingly, we were able to show that heparin binding domain (HBD), the N-terminal portion of TSP-1, was cleaved and secreted simultaneously in a caspase- and serine protease- dependent manner. In the current study we were interested to examine the role of HBD in the clearance of apoptotic cells, and whether the phagocytic and tolerizing state of DCs is mediated by the HBD itself, or whether the entire TSP-1 is needed. Therefore, we have cloned the human HBD, and compared its interactions with DC to those with TSP-1. Here we show that rHBD by itself is not directly responsible for immune paralysis and tolerizing phenotype of DCs, at least in the monomeric form, but has a significant role in rendering DCs phagocytic. Binding of TSP-1-C-terminal domain on the other hand induces a tolerizing phenotype in dendritic cells.

Altered dendritic cells with tolerizing phenotype in patients with systemic lupus erythematosus.

Earlier we showed the generation of tolerizing human monocyte-derived DC following interaction with iC3b-opsonized apoptotic cells. In this study we examine the generation of DC with our previously described tolerogenic phenotype in patients with the systemic autoimmune disease systemic lupus erythematosus (SLE). Monocyte-derived DC were generated in 71 SLE patients, characterized, and then tested for clearance of iC3b-opsonized 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanineperchlorate-stained apoptotic cells using flow cytometry, and for autologous T-cell activation using autologous mixed lymphocyte reaction (AMLR), at the same time as controls. Compared with healthy, age- and gender-matched controls, SLE patients showed upregulation of MHC class II, with a mean expression of 130.5%+/-36.8% (p < 0.007); CD86 in immature DC from SLE patients, generated in autologous human or control plasma, were also upregulated, with mean expression 106.6%+/-18.0% (p < 0.03). A significant (> 20%) reduction in iC3b-apoptotic cell uptake, together with increased autologous mixed lymphocyte reaction, was seen in 75% of SLE patients. Mean 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanineperchlorate-stained apoptotic cell acquisition was 70.0%+/-24% (p < 0.0001) compared with healthy controls. Altered generation of a tolerizing DC phenotype was seen in at least one third of SLE patients following interaction with iC3b-opsonized apoptotic cells. These results suggest that a substantial portion of SLE patients fail to generate DC with a tolerizing phenotype.

Calcium, leukocyte cell death and the use of annexin V: fatal encounters.

One hallmark of programmed cell death (PCD) is redistribution of phosphatidylserine (PS) to the plasma membrane's outer leaflet. Annexin V is widely used in cell death research due to its calcium-dependent ability to bind phosphatidylserine, thus marking apoptotic cells. However, calcium is invariably used at high concentrations in annexin V staining, at doses that can induce cell death. We used flow cytometric annexin V staining, together with propidium iodide and TMRM for determination of dissipation of mitochondrial potential, with a variety of calcium concentrations, cell media, and incubation times, to identify a possible bias in PCD determination of human primary leukocytes. Here we show that measurements of PCD in human monocytes, polymorphonuclear cells, and monocyte-derived dendritic cells using annexin V may be dramatically affected by calcium concentration, time of incubation on ice, and media choice. We propose a method that enables accurate and unbiased annexin V staining, without affecting results.

Apoptotic cell thrombospondin-1 and heparin-binding domain lead to dendritic-cell phagocytic and tolerizing states.

Apoptotic cells were shown to induce dendritic cell immune tolerance. We applied a proteomic approach to identify molecules that are secreted from apoptotic monocytes, and thus may mediate engulfment and immune suppression. Supernatants of monocytes undergoing apoptosis were collected and compared using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and differentially expressed proteins were identified using tandem mass spectrometry. Thrombospondin-1 (TSP-1) and its cleaved 26-kDa heparin-binding domain (HBD) were identified. We show that TSP-1 is expressed upon induction of monocyte apoptosis in a caspase-dependent pattern and the HBD is cleaved by chymotrypsin-like serine protease. We further show that CD29, CD36, CD47, CD51, and CD91 simultaneously participate in engulfment induction and generation of an immature dendritic cell (iDC) tolerogenic and phagocytic state. We conclude that apoptotic cell TSP-1, and notably its HBD, creates a signalosome in iDCs to improve engulfment and to tolerate engulfed material prior to the interaction with apoptotic cells.

Translocation of active heparanase to cell surface regulates degradation of extracellular matrix heparan sulfate upon transmigration of mature monocyte-derived dendritic cells.

After Ag capture and exposure to danger stimuli, maturing dendritic cells (DCs) migrate to regional lymph nodes, where antigenic peptides are presented to T lymphocytes. To migrate from peripheral tissue such as the epidermis to regional lymph nodes, Ag-bearing epidermal Langerhans cells must move through an extracellular matrix (ECM) of various compositions. The nature of their capacity to transmigrate via ECM is not well understood, although MIP-3beta and CCR7 play critical roles. We were interested in verifying whether heparanase, a heparan sulfate-degrading endo-beta-d-glucuronidase that participates in ECM degradation and remodeling, is expressed and functional in monocyte-derived DCs. Using immunohistochemistry, confocal microscopy, RT-PCR, Western blot analysis, assays for heparanase activity, and Matrigel transmigration, we show that heparanase is expressed in both nuclei and cytoplasm of immature DCs, and that gene expression and synthesis take place mainly in monocytes and early immature DCs. We also found that both nuclear and cytoplasm fractions show heparanase activity, and upon LPS-induced maturation, heparanase translocates to the cell surface and degrades ECM heparan sulfate. Matrigel transmigration assays showed a MIP-3beta-comparable role for heparanase. Because heparan sulfate glycosaminoglycans play a key role in the self-assembly, insolubility, and barrier properties of the ECM, the results of this study suggest that heparanase is a key enzyme in DC transmigration through the ECM.

The efficiency of B cell receptor (BCR) editing is dependent on BCR light chain rearrangement status.

Anti-DNA knock-in mice serve as models for studying B cell tolerance mechanisms to a ubiquitous antigen. We have constructed six strains of double transgenic (C57BL/6xBALB/c)F1 mice, each expressing an unmutated or somatically mutated anti-DNA heavy (H) chain, combined with one of three different light (L) chains, namely V(kappa)1-J(kappa)1, V(kappa)4-J(kappa)4 and V(kappa)8-J(kappa)5. In vitro analysis of the various Ig H/L chain combinations showed that all had a similar specificity for single-stranded DNA and double-stranded DNA, but that antibodies encoded by the mutated H chain had higher affinities for the autoantigen. None of the targeted mouse strains exhibited significant levels of serum anti-DNA activity. However, while B cells from mice carrying the V(kappa)1-J(kappa)1 transgenic L chains were tolerized almost exclusively by L chain receptor editing in an affinity-independent manner, the mice expressing V(kappa)8-J(kappa)5 L chains have utilized affinity-dependent clonal anergy as their sole mechanism of B cell tolerance. V(kappa)4-J(kappa)4 targeted mice exhibited an intermediate phenotype with respect to these two mechanisms of B cell tolerance. Our results suggest that receptor editing is the preferred mechanism of B cell tolerance and that the efficiency of L chain editing is directly related to the number of available J(kappa) segments on the expressed V(kappa) allele.